The objective is to highlight the influence of the microstructure of quenched and tempered martensitic steels on the diffusion and trapping of hydrogen. Different steels were compared in term of microstructural parameters. Electrochemical permeation permits to define the parameters that describe the diffusion and trapping of hydrogen in martensitic steels using trapping models. Three hydrogen states have been distinguished: hydrogen lattice concentration, hydrogen reversible trapping sites associated with elastic field and irreversible trapping sites associated with dislocation core and/or vacancy. Nature and energy associated to trapping sites are discussed in relation with the steel microstructure. Thermal desorption spectroscopy (TDS) was used to evaluate the concentration of trapped hydrogen and the nature of the sites implied in the trapping of hydrogen. The influence of a mechanical state was evaluated and an evolution of the diffusion mechanism was observed in the different mechanical states in correlation with the evolution of the microstructure.